Mir-302 is the most predominant microRNA (miRNA) found in human embryonic stem (hES) and induced pluripotent stem (iPS) cells, yet its function is unclear. Previous studies have shown that ectopic expression of mir-302 is able to reprogram human cancer cells to hES-like pluripotent cells with a distinct slow cell cycle rate and dormant cell-like morphology (Lin et al., 2008). Relative quiescence is a defined characteristic of these mir-302-reprogrammed pluripotent stem (mirPS) cells, while other three/four factors (i.e. Oct4-Sox2-Klf4-c-Myc or Oct4-Sox2-Nanog-Lin28)-induced pluripotent stem (iPS) cells have dramatic proliferative ability and inexorable tumorigenetic tendency (Takahashi et al., 2006; Yu et al., 2007; Wernig et al., 2007). Despite the mechanism underlying this anti-proliferative characteristic of mirPS cells is largely unknown, we have identified the possible involvement of two mir-302-targeted G1-checkpoint regulators, cyclin-dependent kinase 2 (CDK2) and cyclin D (Lin et al., 2008). Progression in the eukaryotic cell cycle is driven by activities of cyclin-dependent kinases (CDKs), which forms functional complexes with positive regulatory subunits, cyclins, as well as by negative regulators, CDK inhibitors (CKIs, such as p14/p19Arf, p15Ink4b, p16Ink4a, p18Ink4c, p21Cip1/Waf1, and p27Kip1). In mammalian cells, different cyclin-CDK complexes are involved in regulating different cell cycle transitions, such as cyclin-D-CDK4/6 for G1 progression, cyclin-E-CDK2 for G1-S transition, cyclin-A-CDK2 for S-phase progression, and cyclinA/B-CDC2 (cyclin-A/B-CDK1) for entry into M-phase. Thus, it is conceivable that the anti-proliferative function of mir-302 may result from the co-suppression of CDK2 and cyclin D during G1-S transition.
However, studies of the mir-291/294/295 family, an analog to human mir-302 in mouse, revealed a totally different result from the mir-302 function in human mirPS cells. In mouse embryonic stem (mES) cells, ectopic expression of mir-291/294/295 promoted fast cell proliferation and G1-S transition through direct silencing of p21Cip1 (also named CDKN1A) and serine/threonine-protein kinase Lats2 (Wang et al., 2008). This tumor-prone result was presumed due to the tumor suppressor nature of p21Cip1 and Lats2. Transgenic mice lacking p21Cip1/Waf1 were shown to display normal development with a defect in the G1 checkpoint control (Deng et al., 1995). Yet, the role of Lats2 remains to be determined because of its function in recruitment of amma-tubulin and spindle formation at the onset of mitosis. Loss of Lats2 in mouse embryos was found to cause severe mitotic defects and lethality, indicating that silencing of Lats2 may hinder rather than facilitate cell division (Yabuta et al., 2007). Taken together, silencing of p21Cip1 seems to be the key mechanism underlying such mir-291/294/295-induced tumorigenecity. Nevertheless, our recent effort to screen the mir-302 target site in human p21Cip1 gene shows negative. The same negative result was also predicted by online computing programs TARGETSCAN and PICTAR-VERT. Therefore, mir-302 and its analog mir-291/294/295 likely have different functions in hES and mES cells, lending different characteristics to human and mouse iPS cells. This finding suggests that the role of mir-291/294/295 in mES cells cannot serve as an equivalent model for evaluating mir-302 function in hES and iPS cells.
MiRNA is a cytoplasmic inhibitor and often functions to suppress the translation of it targeted gene transcripts (mRNAs) with high complementarity. The binding stringency between miRNA and its target genes determines the real function of a miRNA. Depending on the cellular condition, miRNA may present different preferences in gene targeting. However, there is no report related to either the concentration effect of mir-302 or the stringency of mir-302-target gene interaction. To resolve this problem, our present invention provides insight into these important details and for the first time reveals that mir-302 functions very differently in human and mouse cells. In humans, mir-302 strongly targets CDK2, cyclins D1/D2 and BMI-1, but interestingly, not p21Cip1. Unlike mouse p21Cip1, human p21Cip1 does not contain any target site for mir-302. This different gene targeting leads to a significant schism between respective cell cycle regulations. In mES cells, mir-302 silences p21Cip1 and promotes tumor-like cell proliferation (Wang et al., 2008; Judson, 2009), whereas p21Cip1 expression is preserved in human mirPS cells and may cause slower cell proliferation and lower tumorigenecity. Additionally, mouse BMI-1 is not a target gene for mir-302 either due to lack of a proper target site. We have found that silencing of human BMI-1 in human mirPS cells stimulates p16Ink4a/p14ARF expression to attenuate cell proliferation, whereas mir-302 cannot silence mouse BMI-1 to raise the same effect in mouse cells. Since p16Ink4a/p14ARF are elevated while p21Cip1 is not affected in mirPS cells, the anti-proliferative function of mir-302 in human cells most likely goes through p16Ink4a-Rb and/or p14/19ARF-p53 pathways in addition to the co-suppression of cyclin-E-CDK2 and cyclin-D-CDK4/6 pathways. These distinct targeting preferences of mir-302 to human and mouse genes imply that the mechanisms underlying their cell cycle regulations are fundamentally different in human and mouse cells.
In sum, prior arts overlooked the stringency of miRNA-target gene interaction and thus misled human mir-302 function into a wrong assumption. To clarify this misunderstanding, our present invention adopted an inducible mir-302 expression system to reveal a novel function of mir-302 in inhibition of human tumor/cancer cell growth, of which our new finding is useful for developing universal anti-tumor/cancer drugs and/or vaccines for cancer therapy as well as prevention. Therefore, there remains a need for effective and safe designs and methods for utilizing mir-302 and its precursors as well as homologues/derivatives in drug/vaccine development and cancer therapy.